“fragmentome” mapping reveals novel, domain-specific partners

Quantitative “Fragmentome” Mapping RevealsNovel, Domain-specific Partners for theModular Protein RepoMan (Recruits PP1 OntoMitotic Chromatin at Anaphase)*!S

Michele Prevost‡, Delphine Chamousset‡, Isha Nasa§, Emily Freele‡, Nick Morrice¶,Greg Moorhead§, and Laura Trinkle-Mulcahy‡!

RepoMan is a protein phosphatase 1 (PP1) regulatorysubunit that targets the phosphatase to key substratesthroughout the cell cycle. Most work to date has focusedon the mitotic roles of RepoMan/PP1, although equallyimportant interphase role(s) have been demonstrated. Ini-tial mapping of the interactome of nuclear RepoMan, bothendogenous and tagged, was complicated by various fac-tors, including antibody cross-reactivity and low sensitiv-ity of the detection of chromatin-associated partnersabove the high background of proteins that bind nonspe-cifically to affinity matrices. We therefore adapted thepowerful combination of fluorescence imaging with label-ing-based quantitative proteomics to map the “fragmen-tomes” of specific regions of RepoMan. These regionsdemonstrate distinct localization patterns and turnoverdynamics that reflect underlying binding events. The in-creased sensitivity and signal-to-noise ratio provided bythis unique approach facilitated identification of a largenumber of novel RepoMan interactors, several of whichwere rigorously validated in follow-up experiments, in-cluding the association of RepoMan/PP1 with a specificPP2A-B56! complex, interaction with ribosomal proteinsand import factors involved in their nucleocytoplasmictransport and interaction with proteins involved in theresponse to DNA damage. This same strategy can beused to investigate the cellular roles of other modularproteins. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.023291, 1468–1486, 2013.

Reversible protein phosphorylation is the major generalmechanism regulating most physiological processes in eu-karyotic cells, with protein kinases and protein phosphatasesplaying key roles in the control of cell proliferation, differenti-

ation, and a host of other critical events. A common theme inphosphatase regulation is a mechanism whereby localizationof the enzyme determines its access to substrates. In the caseof the ubiquitous serine/threonine protein phosphatase 1(PP1)1, this is mediated by interaction of the catalytic subunitwith a panel of regulatory proteins termed “targeting sub-units” to generate a range of holoenzyme complexes withdistinct subcellular roles. Using a novel combination of livecell imaging with Stable Isotope Labeling by Amino acids incell Culture (SILAC)-based quantitative proteomics, we iden-tified several known and novel nuclear PP1 targeting subunitsand determined their specificity for two of the three mamma-lian PP1 isoforms, ! and " (1). We further demonstrated thatone of the novel "-specific interactors, RepoMan, is a target-ing subunit that recruits a pool of PP1 to chromatin initially atanaphase (Recruits PP1 to Mitotic chromatin at ANaphase)and retains it there throughout the following interphase untilits release at the entry into mitosis.

Not surprisingly, this anaphase recruitment of a pool ofRepoMan/PP1 has since been linked to regulation of severalmitotic exit events, including chromosome segregation (2, 3),chromosomal Aurora B kinase targeting (4) and nuclear enve-lope reassembly (5). Persistence on chromatin throughoutinterphase suggests equally important nonmitotic roles forthis phosphatase complex, however. PP1 is known to func-tion in regulation of transcription and chromatin remodeling(for review see 6, 7), and RepoMan/PP1 may therefore con-tribute to modulation of the activity or localization of specifictranscription factors, or chromatin accessibility in general.Consistent with our initial observation that RNA interference-induced knockdown of RepoMan in HeLa cells causes large-scale cell death by apoptosis (i.e., the protein promotes cell

From the ‡Department of Cellular and Molecular Medicine andOttawa Institute of Systems Biology, University of Ottawa, Ottawa,Ontario, Canada; §Department of Biological Sciences, University ofCalgary, Calgary, Alberta, Canada; ¶The Beatson Institute for CancerResearch, Glasgow, Scotland

Received August 27, 2012, and in revised form, December 28, 2012Published, MCP Papers in Press, January 29, 2013, DOI 10.1074/

mcp.M112.023291

1 The abbreviations used are: PP1, protein phosphatase 1; AP/MS,affinity purification/mass spectrometry; FLIP, fluorescence loss inphotobleaching; FRAP, fluorescence recovery after photobleaching;GFP, green fluorescent protein; IP/WB, immunoprecipitation/westernblot; mCh, mCherry; NES, nuclear export signal; NLS, nuclear local-ization signal; NoLS, nucleolar localization signal; SILAC, stable iso-tope labeling by amino acids in cell culture; STLC, S-trityl-L-cysteine.

Technological Innovation and Resources© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This paper is available on line at http://www.mcponline.org

1468 Molecular & Cellular Proteomics 12.5

survival;1), it was recently shown that increased levels ofcellular RepoMan increase the activation threshold of a DNAdamage checkpoint, whereas knockdown restores normalDNA damage response (8).

To identify binding partners for RepoMan that would pro-vide clues to the specific chromatin-associated regulatorypathway(s) in which this PP1 complex is involved, we initiallyapplied our optimized quantitative labeling-based affinity pu-rification/mass spectrometry (AP/MS) approach (9, 10) to as-sess the interactome of nuclear fluorescence protein (FP)-tagged RepoMan. We identified and validated several knownand novel interactors, however none provided obvious links tothe interphase chromatin association of RepoMan. The SILACisotope labeling strategy, with its built-in negative control,provides great help in separating specific interactors fromnonspecific interactors, of which the latter can account for upto 95% of identified proteins in an AP/MS experiment (11, 9).However, not all specific interactions can be unambiguouslydetermined, particularly near the threshold level where signal-to-noise ratios are close to background. A large number ofchromatin-associated proteins bind nonspecifically to affinitymatrices (9), reducing the ability to distinguish genuineprotein–protein interactions. We therefore needed a way toincrease the signal, that is, the abundance of chromatin-associated RepoMan complexes.

We show here that photokinetic analyses of FP-taggedRepoMan and truncated fragments spanning predicted glob-ular domains in the protein provides important clues to thespecific regions involved both in chromatin association and inassociation with other nuclear structures and complexes.Fragments with overlapping regions show similar localizationpatterns and turnover dynamics, reflecting conserved under-lying binding events. When we extended these analyses tointeractome mapping in a quantitative “fragmentome” ap-proach, we identified a large number of previously undetectedRepoMan interactions mediated by distinct regions within thismodular protein. They include the first complex detected be-tween a PP1 holoenzyme (RepoMan/PP1) and a PP2A ho-loenzyme (PP2Ac/R1A/B56!), association of RepoMan withribosomal proteins and import factors involved in their nucle-ocytoplasmic trafficking and an interaction between RepoManand the catalytic subunit of the DNA damage response proteinkinase DNA-PK. With the exception of 14-3-3 proteins, whichmay represent a cell cycle- or perturbation-specific interaction,all of these associations, uncovered by our unique fragmentomeapproach, validated with full-length RepoMan. This confirms theincreased sensitivity of the fragmentome approach and sug-gests previously unknown roles for RepoMan in a diverse rangeof intracellular pathways.

EXPERIMENTAL PROCEDURES

Cloning and Plasmid Constructs—Previously described EGFP- andmCherry (mCh)-tagged RepoMan constructs (1) were used as PCRtemplates to create the RepoMan truncation mutants used in this

study, a full list of which can be found in supplemental Table S1.Phospho-mimic (Ser/Thr to Asp/Glu) and nonphosphorylatable (Ser/Thr to Ala) mutants were generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). EGFP-RPL27,EGFP-RPL5, and GFP-RPS6 were described previously (12), as waspEYFP-NLS (13). mCh-KPNA2 and mCh-KPNB1 were generous giftsfrom Dr. Saskia Hutten, EGFP-Caprin from Dr. Ben Wang and the R1Acoding sequence from Dr. Anne-Claude Gingras.

Cell Culture—HeLa cells (ATCC, Manassas, VA) were grown inDulbecco’s modified Eagles’ medium (DMEM) supplemented with10% fetal bovine serum and 100 U/ml penicillin and streptomycin(Wisent, St. Bruno, QC). HeLa/EGFP, HeLa/EGFP-PP1", and HeLa/EGFP-PP1! stable cell lines were described previously (1). Cells weretransiently transfected using either the Effectene transfection reagent(Qiagen, Valencia, CA) or polyethylenimine (Polysciences, Inc., War-rington, PA). FACS analyses were performed as previously described(14). For mitotic arrest experiments, cells were treated with either 0.1#g/ml nocodazole, 10 nM taxol, or 5 #M S-trityl-L-cysteine (STLC) for18 h before harvesting by mitotic shake-off. S-phase arrest treat-ments included: 0.5 mM L-mimosine for 22 h, 2 mM hydroxyurea for16 h (which was then removed for 2 h before harvest by changing themedia) or a double thymidine block in which cells were treated with 2mM thymidine for 14 h, the thymidine removed for 18 h and then 2 mM

thymidine added for another 14 h before harvest.Fluorescence Imaging and Photokinetic Experiments—Live cell im-

aging experiments were carried out as described previously, using awide-field deconvolution-based fluorescence microscope system(DeltaVision CORE; Applied Precision, Issaquah, WA) equipped with athree-dimensional motorized stage, temperature-and gas-controlledenvironmental chamber, Xenon light source, and quantifiable lasermodule (13). Images were collected using a 60 ! NA 1.4 Plan-Apochromat objective and recorded with a CoolSNAP coupled-charge device (CCD) camera (Roper Scientific, Trenton, NJ). Themicroscope was controlled by SoftWorX acquisition and deconvolu-tion software (Applied Precision, Seattle, WA). For the permeabiliza-tion assay, cells were imaged before and after 5 min incubation with0.1% Triton X-100 in ASE buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.5mM EGTA) at 37 °C.

For FRAP experiments, a single section was imaged before pho-tobleaching, a region of interest (ROI) bleached to 50–60% of itsoriginal intensity using a 488-nm laser, and a series of images ac-quired after the photobleaching period. Images were analyzed andrecovery half-times and mobile fractions calculated using SoftWorXor GraphPad Prism. Recovery curves were plotted using MicrosoftExcel. For FLIP experiments, different areas of the cell were moni-tored while a specific region was repeatedly photobleached overtime.

Cell Extracts and Immunoblotting—Whole-cell extracts and cyto-plasmic, nucleoplasmic and nucleolar fractions were prepared aspreviously described (9, 15). The final buffer for each extract was RIPA(50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxy-cholate, and protease inhibitors), and all extracts were cleared bycentrifuging at 2800 ! g for 10 min at 4 °C. Nuclear extracts wereprepared from purified nuclei as previously described (1). Total pro-tein concentrations were measured using a Qubit Fluorometer (Invit-rogen, Carlsbad, CA) or Pierce® BCA Protein Assay Kit (ThermoScientific, Rockford, IL).

For affinity purification of GFP- and mCh-tagged proteins, extractswere incubated with either GFP-Trap_A™ or RFP-Trap_A™, respec-tively (Chromotek, Martinsried, Germany). For immunoprecipitation(IP) of endogenous proteins, affinity purified antibodies were cova-lently conjugated to protein G Sepharose (GE Healthcare, Missis-sauga, ON) at a concentration of 1 mg/ml. Covalently-conjugatedaffinity purified IgG from the same species was used for control IPs.

Domain-specific RepoMan Interactome

Molecular & Cellular Proteomics 12.5 1469

Beads were incubated with extracts for 1 h at 4 °C, after which theextracts were removed, the beads washed several times with ice-coldRIPA buffer and bound proteins eluted by heating in LDS samplebuffer (Invitrogen). Lysate and IP samples were separated on 4–12%Novex Nu-PAGE bis-Tris polyacrylamide gels (Invitrogen) and trans-ferred to nitrocellulose membranes for immunoblotting.

To demonstrate the mobility shift of the phosphorylated proteins,Phos-tag™ ligand (final concentration 50 !M) and MnCl2 (final con-centration 100 !M) were added to the 10% polyacrylamide separatinggels before polymerization. After electrophoresis the gel was soakedin 2 mM EDTA, 25 mM Tris, and 192 mM glycine for 15 min to eliminatemanganese ions from the gel and then equilibrated in transfer buffer(25 mM Tris,192 mM glycine, and 20% methanol) for another 15 min.Proteins were transferred to nitrocellulose membranes at 4 °C for400 Vh.

A full list of all of the primary antibodies used for immunoblottingcan be found in supplemental Table S2. To reduce the antibodyheavy chain signal and improve resolution of the B56" band, theReliaBLOT® Western blot kit (Bethyl Laboratories, Inc., Montgomery,TX) was used according to the manufacturer’s instructions. Primaryantibodies were detected via HRP-conjugated secondary antibodies(Thermo Scientific) and ECL Plus Western blotting substrate (GEHealthcare). Full Western blots are provided (Supplemental Figs. S6–S8) for all cropped blots presented in Figs. 1, 4, 5, 6 and 7.

Quantitative SILAC IP—Quantitative SILAC-based affinity purifica-tion experiments were carried out as previously described (9). Cellswere encoded in media containing either “light” L-arginine and L-lysine (Sigma-Aldrich, Oakville, ON), “medium” L-arginine 13C andL-lysine 4,4,5,5-D4 or “heavy” L-arginine 13C/15N and L-lysine 13C/15N(Cambridge Isotope Laboratories, Andover, MA) and extracts pre-pared as described above. Control (tag alone) and fusion proteinpurifications were carried out separately, the extracts removed andthe beads washed once with ice-cold RIPA buffer, and then thecontrol and experimental beads carefully combined and washed anadditional three times with ice-cold RIPA buffer. Proteins were eluted,separated by 1D SDS-PAGE, and trypsin-digested for MS analysis asdescribed previously (9).

Mass Spectrometry and Data Analysis—An aliquot of the trypticdigest (prepared in 5% acetonitrile/0.1% trifluoroacetic acid in water)was analyzed by LC-MS on an LTQ-Orbitrap mass spectrometersystem (ThermoElectron, Rockford, IL) as previously described (9).The Orbitrap was set to analyze the survey scans at 60,000 resolutionand the top five ions in each duty cycle selected for MS/MS in the LTQlinear ion trap. Database searching (against the human IPI databasev3.68; 87,061 entries) and quantitation were performed using MaxQuantsoftware v1.1.1.14 and the Andromeda search engine (16, 17). Thefollowing criteria were used: peptide tolerance ! 10 ppm, trypsin asthe enzyme (2 missed cleavages allowed) and carboxyamidomethy-lation of cysteine as a fixed modification. Variable modifications wereoxidation of methionine and N-terminal acetylation. Medium SILAClabels were Arg6 and Lys4 and heavy SILAC labels were Arg10 andLys8. Minimum ratio count was 2 and quantitation was based on razorand unique peptides. Peptide and protein FDR was 0.01. The Perseusdata mining and management module of MaxQuant was used tocalculate Significance B values. For quantitation of specific PP1 iso-forms and RepoMan fragments, only unique SILAC ratios were aver-aged. All data sets (minus common environmental contaminants asper http://maxquant.org and proteins identified via the decoy data-base) are provided as Supplemental Data.

Phosphopeptide Mapping—For phosphopeptide mapping ofRepoMan in nocodazole-arrested HeLa cells, the endogenous proteinwas immunopurified from cell extracts using a covalently conjugatedpolyclonal RepoMan antibody (Abcam, Cambridge, MA). Proteinswere separated by SDS-PAGE and the region containing the phos-

phorylated RepoMan band was excised from the gel (as determinedby alignment with a Western blot run concurrently to highlight theposition of phospho-RepoMan). The excised band was incubatedwith 50 mM iodoacetamide to alkylate cysteine residues, washed anddigested with trypsin for 16 h. Tryptic digests were analyzed byLC-MS/MS using a Proxeon Easy-LC connected to an LTQ orbitrapvelos system via a Proxeon nanospray source fitted with a NewObjective FS360–20-20 uncoated emitter. An aliquot of 18/100 !l ofthe tryptic digest was injected onto 20 " 0.1 mm C18 guard columnequilibrated in buffer A (2% acetonitrile/0.1% formic acid in water) at7 !l/min. After washing the injector loop with 30 !l buffer A, the guardcolumn was switched in line with a 150 " 0.075 mm PepMap C18column (Dionex) equilibrated in buffer A at 300 nl/min. The columnwas developed with a 60 min discontinuous gradient of buffer B (80%acetonitrile/0.1% formic acid in water). Nanoelectrospray was per-formed by applying a voltage of 1.6 kV to the emitter and the orbitrapwas set to perform a survey scan of m/z 350–1800 at a resolution of60000 and the top 10 multiply charged ions (minimum intensity 10000cps) were selected for collision induced dissociation in the LTQ andthen excluded for 30 s after two occurrences. The raw data file wasconverted to a Mascot Generic File (mgf) and searched using Mascot2.4 and Proteome Discoverer 1.3. The following criteria were applied:Database, SwissProt; Species, Human;Enzyme, Trypsin (1 missedcleavage permitted); Fixed modification, Carboxyamidomethylation ofcysteine; Variable Modification, Oxidation of Methionine, phosphory-lation (STY), Gln#pyroglu; Precursor mass tolerance, 10 ppm,MS/MS mass tolerance, 0.8 Da. Phosphopeptides were assignedusing phosphoRS and validated by searching a reverse database withthe FDR set at 0.01 (Strict) and 0.05 (relaxed) Supplemental Fig. S9.

RESULTS

Quantitative SILAC IP Identifies Novel RepoMan Com-plexes—RepoMan is predominantly nuclear in interphasecells (1), and we therefore combined our quantitative SILAC IPapproach (9) with cellular fractionation to reduce backgroundcontaminants and increase sensitivity of our nuclear RepoManinteractome mapping. Two sets of isotopic labels (R6K4 andR10K8) were used to directly compare the wild type RepoManinteractome to that of a non-PP1 binding mutant (RepoMan/RAXA;1), to determine whether the ability to recruit PP1 af-fects the molecular complexes with which RepoMan associ-ates. Isotopically labeled HeLa cells were transientlytransfected with either mCh alone (control) or mCh-taggedRepoMan fusion proteins, harvested and fractionated intocytoplasm and nuclei. Pull-down experiments were per-formed on nuclear extracts using the red fluorescent protein(RFP)-Trap_A™ affinity matrix and combined before massspectrometric analysis (Fig. 1A).

As shown in the graph in Fig. 1B, although the majority ofidentified proteins represent common bead contaminants, asexpected (9), a subset was enriched with tagged RepoMan.This group includes both known and novel interactors. Withthe exception of the PP1 isoforms, all of the detected inter-actors were enriched equally with wild type and mutantRepoMan. Fig. 1C summarizes the top hits (log2 M: L ratios #1), all of which were identified by two or more unique peptidesand enriched significantly above background (see Supple-mental Data for full data set). Proteins shown in bold werevalidated as bona fide RepoMan interactors. Although the



FIG. 1. Quantitative analysis of the tagged RepoMan interactome. A, Design of the quantitative IP experiment comparing mCh-RepoMan(M), the non-PP1 binding mCh-RepoMan/RAXA mutant (H), and the mCh tag alone (L). B, Plot of log H:L ratio (enrichment in mCh-RM/RAXAIP) versus log M/L ratio (enrichment in mCh-RM IP). Contaminants cluster around a log ratio 0 (SILAC ratio 1:1), whereas proteins enrichedequally with both fall in the upper right quadrant. C, Table listing # peptides (total and unique), ratio M/L (enrichment with mCh-RepoMan), ratioH/L (enrichment with mCh-RepoMan/RAXA) and ratio H/M (enrichment with mutant relative to wild type) for proteins with M/L ratios ! 2identified with two or more peptides. Significance B (Sig B) values, which are weighted by relative intensity, are shown for those proteins withratios significantly different from the mean (p " 0.05). Proteins highlighted in bold have been validated as bona fide RepoMan interactors byIP/WB analysis. D, Validation of the copurification of KPNB1 and KPNA2 with tagged, full-length RepoMan (RM) from asynchronous cellextracts. E, Co-IP/WB validation of novel interactions of RepoMan with two additional importins, KPNA4 and IPO7. F, Copurification ofPPP2R1A with FP-tagged wild type and RAXA mutant RepoMan. G, Reciprocal validation of the R1A/RepoMan interaction demonstrated bycopurification of endogenous RepoMan with FP-tagged R1A. As a control, PP2Ac was also shown to copurify with FP-R1A. Anti-FP antibodiesdemonstrate the amount of fusion protein (or free FP) recovered in each IP.



protein arginine N-methyltransferase PRMT1 appeared en-riched with both wild type and mutant RepoMan in the SILACexperiment, we were unable to convincingly validate this in-teraction by IP/WB analysis (data not shown).

Previously identified interactors include NUP153, KPNA2,and KPNB1, believed to link RepoMan to regulation of nuclearenvelope formation at mitotic exit (5). We confirmed interac-tion of KPNA2 and KPNB1 with FP-RepoMan in asynchro-nous HeLa cell extracts by immunoprecipitation and Westernblot (IP/WB) analysis (Fig. 1D), and demonstrated that FP-tagged KPNA2 and KPNB1 copurify endogenous RepoMan(supplemental Fig. S4D). We also identified and validatednovel interactions between FP-RepoMan and two additionalimportins, KPNA4 and IPO7 (Fig.1E).

A surprising result was enrichment of the PP2A regulatorysubunit R1A (a.k.a. PR65A, !-isoform of the 65 kDa scaffold-ing A subunit), which suggests association of a PP1 holoen-zyme complex with a PP2A holoenzyme complex. To validatethis interaction, we first demonstrated copurification of en-dogenous R1A (and the PP2A catalytic subunit PP2Ac) withtagged RepoMan (wild type and RAXA mutant) by IP/WBanalysis (Fig. 1F). We then cloned R1A and demonstratedcopurification of endogenous RepoMan (and PP2Ac) withtagged R1A (Fig. 1G). This confirms that RepoMan can com-plex with the core PP2Ac/R1A holoenzyme. No PP2A regula-tory B subunits were identified in the SILAC screen.

Fluorescence Imaging-based Assays of GFP-taggedRepoMan Fragments Reveal Distinct Subnuclear Localizationand Binding Affinity Regions within the Full-Length Protein—Although our quantitative IP analysis of tagged full-lengthRepoMan identified several interaction partners, there was adistinct absence of chromatin-associated proteins. RepoManis recruited to and persists on chromatin during mitotic exit,and its localization and dynamics in interphase are consistentwith maintenance of this chromatin association (1). BecauseRepoMan itself does not contain any known DNA bindingdomains, it is assumed that this association is indirect, withRepoMan binding chromatin-associated proteins. The pre-dominance of importins in the SILAC experiment suggestedthat repeated identification of these highly abundant interac-tors may have precluded identification of less abundant bind-ing partners. With evidence that importin " association mapsto the extreme N terminus (5) and chromatin association to theC-terminal half (8), we hypothesized that splitting RepoManinto distinct domains with different underlying binding eventswould increase signal/noise and facilitate identification oflower abundance binding partners. We therefore combined amicroscopy-based characterization of RepoMan truncationmutants with our quantitative proteomic approach in a unique“fragmentome” strategy.

Although the three-dimensional structure of RepoMan hasnot yet been solved, structural algorithms such as GlobPlot(18) can be used to predict likely globular versus disorderedregions within the protein based on amino acid sequence (Fig.

2A). We used these predicted regions to design a RepoManfragment cloning strategy that would maximize proper foldingof the expressed fragments, with all fragments being fused toan N-terminal GFP tag.

Using a range of in vivo fluorescence imaging approaches,we compared the behavior of the various RepoMan fragmentsto that of the full-length protein, starting with analysis oflocalization in live cells. Cells were imaged before and aftermild permeabilization with 0.1% Triton X-100, to comparenuclear retention. Freely soluble proteins are rapidly lost fromthe nucleus in response to this treatment (see GFP and YFP-NLS in Fig. 2A), whereas proteins that form more stableassociations with other nuclear proteins or structures areretained. Binding events also affect the turnover of GFP-tagged proteins in fluorescence recovery after photobleach-ing (FRAP) experiments, in which a pool of GFP-protein isirreversibly photobleached by exposure to high intensity 488nm light and recovery of the fluorescence signal within thisregion of interest (which represents turnover of the protein onbinding sites) monitored over time. Lastly, fluorescence loss inphotobleaching (FLIP) experiments were used to test nuclear/cytoplasmic shuttling. Repeated photobleaching of a regionof interest in the cytoplasm of a cell leads to an eventual lossof nuclear fluorescence if the GFP-tagged protein activelyshuttles or freely distributes between the nucleus and cyto-plasm (and thus passes through the photobleached region atsome point).

Like endogenous RepoMan, GFP-RepoMan is predomi-nantly nuclear in interphase cells, with a localization patternsimilar to that of DNA binding dyes such as DAPI and Hoechst33342 (Fig. 2A,1). The protein contains four predicted nuclearlocalization signals (NLSs) and no nuclear export signals(NESs). Consistent with this, once RepoMan is recruited to thenucleus it remains there and does not shuttle between thenucleus and cytoplasm, as demonstrated by FLIP experi-ments (Fig. 2C). Compared with the rapid loss of free GFPfollowing mild permeabilization of the cells, !80% of GFP-RepoMan is retained in the nucleus (Fig. 2A), reflecting par-ticipation in multiprotein complexes and/or association withnuclear structures.

All of the FP-tagged RepoMan fragments tested here arealso predominantly nuclear, which is not entirely surprisinggiven that nearly all of them contain a predicted NLS (Fig. 2A).Two exceptions are the 1–135 fragment, previously shown tobind directly to KPNB1 in vitro (5), and the C-terminal 713–946fragment, which is likely recruited via association with othernuclear proteins. The majority of the fragments were alsoretained in the nucleus to varying extents following mild per-meabilization, although differences in subnuclear localizationpatterns and turnover dynamics indicate that distinct bindingevents are associated with each of these regions.

When RepoMan was split into N-terminal (1–496) and C-terminal (496–1023) halves, the C-terminal half showed asimilar localization pattern to that of full-length RepoMan,



FIG. 2. Design of truncation mutants for the analysis of domain-specific RepoMan interactomes. A, Localization of GFP-RepoMan(GFP-RM) in live HeLa cells before (“Live” panel) and after (“Perm” panel) mild permeabilization with 0.1% Triton X-100. Localization of free GFPand YFP-NLS (nuclear localization signal added to enhance nuclear and nucleolar accumulation) in the cell is shown for comparison. Scale barsare 5 !M. The diagram in the center shows predicted globular (box) and disordered (line) regions in RepoMan and highlights the PP1-bindingRVxF motif (blue box) and predicted NLSs (red boxes). Localization in live cells before and after mild permeabilization is shown for each



both before and after permeabilization, whereas the N-termi-nal half displayed a diffuse nucleoplasmic pattern and in-creased loss from permeabilized cells. Consistent with theseobservations, the N-terminal 1–496 fragment also demon-strated a more rapid recovery rate in FRAP experiments,whereas the turnover dynamics of the C-terminal half (496–1023) were similar to that of the full-length protein (Figs. 2A,2B). Neither fragment was lost from the nucleus in FLIP ex-periments (Fig. 2C), indicating that they were retained in thenucleus once they were imported. This is consistent with thefact that neither has an export signal nor is small enough to belost by free diffusion through nuclear pores.

Truncated fragments of the N-terminal half of RepoMan allshowed predominantly diffuse nucleoplasmic localization pat-terns, although we did observe colocalization of a small poolof FP-RepoMan (and the 1–305 and 1–496 fragments) withnuclear pore complex markers at the nuclear envelope (datanot shown), consistent with a previous study (5). Thesesmaller N-terminal fragments were not efficiently retained inthe nucleus following permeabilization (Fig. 2A) and showedsimilar and more rapid recovery rates in FRAP experiments(Fig. 2A, supplemental Fig. S1), suggesting that they do formlarger complexes, but that these complexes are predomi-nantly soluble and not as stably associated with nuclear struc-tures as the full-length protein.

Splitting the C-terminal half of RepoMan into two fragments(496–713 and 713–1023) uncovered a surprising nucleolaraccumulation of the 496–713 fragment that mapped to aminimal predicted globular region of 496–591 (Fig. 2A andsupplemental Fig. S5C). This region contains a bipartite NLS,and sequence analysis using the Nucleolar Localization Se-quence Detector (NoD;19) identified a predicted nucleolarlocalization signal (NoLS) overlapping this NLS (Fig. 8B). Al-though full-length RepoMan is predominantly nucleoplasmic,as judged both by imaging of endogenous and GFP-taggedRepoMan in cells and by cell fractionation/WB analysis (1,supplemental Fig. S4A), this simply indicates that there is noaccumulation of the protein in this subnuclear structure understeady-state conditions. FLIP analysis revealed that the nu-clear pool of GFP-RepoMan does in fact shuttle throughnucleoli (supplemental Fig. S5D). The NoLS may therefore beimportant for one or more of RepoMan’s roles within the

nucleus. The 3 fragments that span this NoLS-containingregion (305–713, 496–713 and 496–591) are all retained to agreater extent in the nucleus (and nucleolus) following mildpermeabilization than the N-terminal fragments and also haveslower turnover dynamics, albeit faster than that of full-lengthRepoMan (Figs. 2A, 2B, supplemental Fig. S1).

Although the 713–1023 fragment showed a similarsteady-state nuclear localization pattern to that of full-length RepoMan (and the 496–1023 C-terminal half), it wasnot retained in the nucleus to the same extent following per-meabilization (Fig. 2A) and its turnover dynamics as measuredby FRAP were faster, albeit not as fast as the 1–496 N-ter-minal half or the 496–713 fragment (Figs. 2A, 2B). This high-lights the importance of assessing both localization and dy-namics, as the latter can detect changes in underlying bindingevents that are not necessarily reflected in steady-state local-ization patterns.

Further analysis of the recovery curve of full-length GFP-RepoMan revealed that the data are better fit by a two-phaseassociation consisting of a fast recovery pool (t1/2 ! 1.6 s) anda slow recovery pool (t1/2 ! 18.9 s). This suggests that, understeady-state conditions in interphase nuclei, RepoMan isfound in more than one complex. Our inability to detect chro-matin-associated interactors above the highly abundant im-portins in our intial interactome screen of full-length RepoMan(Fig. 1) may reflect differences in the relative levels of thesecomplexes. We therefore set out to map the “fragmentome”of the C-terminal half expressed on its own, compared it tothat of the dynamic N-terminal half that contains PP1 (1) andimportin ! (5) association domains. Our hypothesis was thatthis reduced complexity, along with the higher expressionlevels that we observe for RepoMan fragments in cells com-pared with the FP-tagged full-length protein (supplementalFig. S2D), should substantially increase our signal-to-noiseratio in interactome experiments.

RepoMan Fragmentome Identifies Distinct Pools of Interac-tors for the N- and C-Terminal Halves of the Protein—We usedour optimized quantitative SILAC IP approach (9) to comparethe fragmentomes of the N-terminal (1–496) and C-terminal(496–1023) halves of RepoMan, as outlined in Fig. 3A. HeLacells were isotopically labeled for 7 days before transientexpression of either GFP alone or the two GFP-tagged

fragment and summarized on the graph as % retained in nucleus. Data are mean " S.E. (n ! 13–40, 3–4 separate experiments). B, FRAP(Fluorescence Recovery After Photobleaching) experiment comparing the recovery rates of the photobleached N-terminal (1–496, greentriangles) and C-terminal (496–1023, purple crosses) halves of RepoMan within a nucleoplasmic region of interest (ROI) to that of the full-lengthprotein (red squares) and free GFP (blue diamonds). Also shown are the recovery curves for the split C-terminal halves: 496–713 (turquoisecrosses) and 713–1023 (orange circles). A pre-bleach image was acquired, a nucleoplasmic ROI photobleached at the 0 time point (arrow) andthe GFP signal within that ROI monitored over time. Data are mean " S.E. (n ! 7–31, three separate experiments). Recovery half-times fornucleoplasmic pools of all fragments tested are summarized in the far right column in (A) as mean " S.E. C, FLIP (Fluorescence Loss InPhotobleaching) experiment assessing nucleocytoplasmic shuttling. A cytoplasmic region of interest was continually photobleached every 1 s(arrows) for a total of 126 s, with images taken after each bleach event. Total intensity over time for a nuclear ROI was normalized forphotobleaching because of image acquisition. Data are mean " S.E. (n ! 4–7, 3 separate experiments). GFP, which shuttles freely betweenthe nucleus and cytoplasm (nuclear pool is continually depleted as the protein trafficks through the cytoplasmic region of interest and isirreversibly photobleached) was used as a positive control.



RepoMan fragments. Because the fragments vary in theirnuclear retention in response to mild permeabilization, wetested their nuclear retention during cell fractionation andfound that a pool of the 1–496 fragment does end up in thecytoplasmic fraction (supplemental Fig. S4C), despite the fu-sion protein being predominantly nuclear in live cells (Fig. 2A;Fig. 4A). For that reason we decided to prepare whole-cellrather than nuclear extracts. As previously noted, we ob-served both an increased transfection efficiency and higherlevel of fusion protein expressed in cells for the fragmentscompared with full-length tagged RepoMan, which was re-flected in a 50% increase in the relative amount of totalprotein recovered in the SILAC IP as judged by relative abun-dance in the MS analysis (supplemental Fig. S3).

The samples were analyzed twice by LC-MS/MS, with atotal of 238 and 277 proteins identified and quantified, re-spectively, in each run (full data sets provided as Supplemen-tal Data). The top hits are summarized both in supplementalTable S3 and in Fig. 4A, which shows a plot of the log H:Lratios (enrichment with 496–1023) versus log M:L ratios (en-richment with 1–496). As expected, PP1 copurified with the1–496 fragment containing the PP1-binding RVxF motif (res-idues 392–395). WB/IP analysis confirmed association ofPP1! both with this fragment and with the 305–496 disor-dered region (Fig. 4B). NUP153, identified in our original nu-clear RepoMan interactome screen (Fig. 1), also copurifiedwith the 1–496 fragment. We confirmed interaction ofNUP153 with both full-length RepoMan and the 1–496 frag-ment by IP/WB, and mapped it to the same minimal 1–135region previously shown to mediate KPNB1 binding (Fig. 4C).

KPNB1, along with the importins KPNA1, A2 and A4, wasalso enriched with the N terminus of RepoMan (Figs. 4A, 4B).Interestingly, the importin IPO7 did not map specifically to thisregion in the proteomic screen, but instead was enrichedequally with both the N- and C-terminal halves of RepoMan(Fig. 4A). We had previously identified and validated IPO7 asa RepoMan interactor in our nuclear interactome screen (Fig.1). IP/WB analysis revealed that efficient copurification ofIPO7 is disrupted by splitting the protein in half, althoughslight enrichment is observed with both halves (fragments1–496 and 496–1023), consistent with the proteomic results(Fig. 4C). This suggests that association of IPO7 withRepoMan involves domains in both the N- and C-terminalhalves of the protein.

Also consistent with its identification in our original full-length RepoMan interactome, the PP2A scaffolding subunitR1A was found to be enriched with the C-terminal fragment,along with the catalytic subunit PP2Ac and the regulatory Bsubunit B56! (Fig. 4A, supplemental Table S3). IP/WB analy-sis confirmed both the association of full-length RepoManwith this PP2A complex (supplemental Fig. S4G) and specificmapping of the interaction to the C-terminal half of RepoMan(Fig. 4D).

FIG. 3. Quantitative mapping of RepoMan “fragmentomes.” A,Design of the quantitative IP experiments comparing the interactomesof RepoMan fragments. B, Distribution of log SILAC (M:L and H:L) ratiosfor the fragmentome experiment comparing GFP-RepoMan/1–496 (M)to GFP-RepoMan/496-1023 (H), with free GFP as the built-in negativecontrol (L). The small peak (arrow) of log H:L ratios (i.e. enriched with496–1023) that separates from the contaminants (peak over log ratio 0,i.e. ratio 1:1) contains enriched ribosomal proteins. C, Distribution of logSILAC (M/L and H/L) ratios for the fragmentome experiment comparingGFP-RepoMan/496–713 (M) to GFP-RepoMan/713–1023 (H), with freeGFP as the built-in negative control (L). The peak (arrow) of log M:Lratios (i.e. enriched with 496–713) that separates from the contaminants(peak over log ratio 0, i.e. ratio 1:1) contains enriched ribosomal proteins.



FIG. 4. N- and C-terminal halves of RepoMan bind distinct pools of interactors. A, Plot of log H:L ratio (enrichment in GFP-RepoMan/496-1023 IP) versus log M:L ratio (enrichment in GFP-RepoMan/1–496 IP). Contaminants cluster around a log ratio 0 (SILAC ratio 1:1). TheN-terminal fragment (blue circle) enriched a cluster of nuclear import factors (yellow triangles) and two isoforms of PP1 (green squares),whereas the C-terminal fragment (red circle) enriched PP2Ac/R1A/B56! (orange squares) and ribosomal proteins (RPLs, dark pink triangles;RPSs, light pink triangles). Histones (turquoise circles) are close to threshold, albeit slightly enriched with the C terminus. Inset images aresingle optical sections of live HeLa cells expressing FP-RepoMan/1–496 or FP-RepoMan/496-1023. Both fragments are predominantlynuclear. Scale bars are 5 "M. B, Validation and further mapping of N-terminally mediated interactions with KPNA2, KPNB1 and PP1. C,Validation of NUP153, IPO7 and RPS6 association with full-length FP-tagged RepoMan (FP-RM). Interaction of NUP153 with the N-terminalhalf of RepoMan was further mapped to the 1–135 domain, whereas RPS6 enrichment was confirmed to be mediated by the C-terminal halfof RepoMan. IPO7 was weakly enriched with both halves and with the 1–135 domain. D, Mapping of the PP2Ac/R1A/B56! association withRepoMan to the C-terminal half of the protein was confirmed by IP/WB analysis. E, Reciprocal validation of the RepoMan/ribosomal proteininteraction was demonstrated by copurification of endogenous RepoMan (RM) with a range of FP-tagged ribosomal proteins (RPL27, RPL5 andRPS6). Anti-FP antibodies were used to demonstrate the amount of fusion protein (or free FP) recovered in each IP. F, Endogenous RPS6,immunoprecipitated using anti-RPS6 antibodies (compared with an IgG control IP), copurifies endogenous RepoMan. RPS6 runs just abovethe 25 kDa antibody light chains (LC), which are indicated (arrow) on the WB.



As shown in Fig. 4A, there was a surprising enrichment of alarge number of ribosomal proteins with the C-terminal half ofRepoMan. Although these are common contaminants knownto bind nonspecifically to affinity matrices (9, 10), genuineenrichment in a quantitative SILAC IP will shift their isotoperatios above those of nonspecific contaminant proteins thatfall in a distribution over a ratio of 1:1 (log ratio 0). Wedemonstrated this previously for the nucleolar proteinRRP1B, which targets PP1 to pre-60S subunits and specif-ically enriches RPL proteins (13). When we plotted the dis-tribution of ratios for the FP-RepoMan/1–496 (log M:L) andFP-RepoMan/496-1023 (log H:L) fragments, we saw a nor-mal distribution for the log M:L ratios, that is, the majority ofproteins are bead contaminants enriched equally under allconditions and thus distributed over a ratio of 1:1 (log ratio0), whereas a small pool of enriched proteins is shifted tohigher ratios. The log H:L ratio distribution, however, in-cluded a substantial “bump” that was shifted to the right (!1:1) of the contaminant ratio peak (Fig. 3B). This groupincludes a large number of ribosomal proteins (supplemen-tal Table S3) that appear to be enriched with the C-terminalhalf of RepoMan.

Given the increased and variable background caused bynonspecific binding of ribosomal proteins in control IPs, en-richment above background must be rigorously and repro-ducibly demonstrated to claim validation of the interaction.We first quantified four separate IP/WB experiments, demon-strating significant enrichment of representative 60S and 40Ssubunit ribosomal proteins (RPL7 and RPS6) with full-lengthFP-RepoMan above the background nonspecific binding ob-served in control FP IPs (supplemental Fig. S5B). Overexpres-sion of RepoMan (or the C-terminal fragment) does not affecttotal cellular levels of ribosomal proteins (data not shown),verifying that the enrichment is specific to the tagged proteinand not simply because of the potential for higher levels ofnonspecific binding to the affinity matrix. We next demon-strated reciprocal copurification of endogenous RepoManwith three different FP-tagged ribosomal proteins (RPL27,RPL5, and RPS6) identified in the fragmentome screen (Fig.4E). Lastly, we demonstrated copurification of endogenousRepoMan with endogenous RPS6 (Fig. 4F).

This stringent approach validates ribosomal proteins asgenuine RepoMan interactors and confirms that the C-termi-nal half of RepoMan mediates the association. Although wehave looked at a subset of ribosomal proteins here, the SILACscreen identified enrichment of a much larger number (Fig.4A, supplemental Table S3, supplemental Fig. S3C), suggest-ing that the interaction is related to a more general role inribosome biogenesis, such as ribosomal protein import orribosome subunit export.

RepoMan Forms a Cell Cycle-dependent Complex withPP2A/R1A/B56!—The identification of a PP2A complex co-purifying with RepoMan was intriguing not only because it isthe first demonstration of an interaction between two specific

PP1 and PP2A holoenzyme complexes, but also becauseRepoMan is predominantly nuclear in interphase cellswhereas PP2Ac, R1A and B56! are predominantly cytoplas-mic (supplemental Fig. S4B). We initially uncovered the inter-action in our nuclear FP-RepoMan SILAC IP, but only R1Awas detected (and identified by only two peptides, Fig. 1C),suggesting that the complex was either at a very low abun-dance in the nucleus or represented trace amounts of cyto-plasmic contamination during fractionation. Identification ofB56! as a member of the complex in the fragmentome ex-periment suggested another possibility. This specific B56 iso-form is one of two that have been proposed to have a nuclearpool (20), and more recently a PP2Ac/B56! complex wasshown to accumulate in nuclei during S phase (21). To test thepossibility of a nuclear, albeit cell cycle-specific, interactionwith RepoMan, we assessed the copurification of B56! withRepoMan in asynchronous cells compared with cells arrestedeither in S phase or in early mitosis. When endogenousRepoMan pulldowns were probed with anti-B56! antibodies,significant enrichment was only observed in cells arrested inearly mitosis (Fig. 5A). We then confirmed the mitosis-specificinteraction of R1A and PP2Ac with RepoMan (Fig. 5B).

In comparison, PP1! is found to associate with RepoManunder both conditions, along with a representative ribosomalprotein (RPL7) and three additional interactors identified in thefragmentome screen (Caprin, G3BP2 and hnRNP K). AlthoughRepoMan is expressed at all stages of the cell cycle, proteinlevels are higher in mitotic cells (see RepoMan lysate lanes inFig. 5B) and consequently more RepoMan is recovered frommitotic cells. When quantified as amount interactor detected/amount RepoMan recovered, most showed only slight differ-ences, however the amount of B56! recovered from nocoda-zole-arrested cells with RepoMan was 2-fold higher than thatrecovered from asynchronous cells. In comparison, the hn-RNP K interaction is more predominant (twofold higheramount recovered) in asynchronous cells.

In parallel with our RepoMan interactome screen, we car-ried out a quantitative comparison of mitotic PP1" and PP1!

complexes, by nocodazole arrest of our HeLa/GFP, HeLa/GFP-PP1" and HeLa/GFP-PP1! stable cell lines. As shown inFig. 5C, we confirmed the specific enrichment of Repomanwith PP1!, and detected significant enrichment of both R1Aand B56! with this isoform. This provides further evidencethat a pool of RepoMan/PP1 associates with a PP2Ac/R1A/B56! complex in early mitosis.

The non-PP1 binding mutant of RepoMan copurifies thecomplex as efficiently as the wild type protein (Figs. 1F, 6E),and we found no evidence that RepoMan/PP1 regulatesphosphorylation of any of the complex members. R1A doesshow a shift in phosphorylation on entry into early mitosis,however, as highlighted by Phos-tag gel/WB analysis of asyn-chronous versus nocodazole (G2/M) or STLC (metaphase)arrested cell extracts (Fig. 5D). The Phos-tag binds phosphategroups on proteins and amplifies the apparent molecular



mass shift (22). In asynchronous cells, FP-RepoMan (and theRAXA mutant) copurify the less abundant, lower (or un-) phos-phorylated form of R1A that likely represents the small pro-portion (!20% of the total) of mitotic cells (Fig. 5E). Interest-ingly, although the 305–713 (contains PP1 binding site) and496–713 (no PP1 binding site) fragments of RepoMan bothcopurify R1A, the specificity for the less phosphorylated formobserved with full-length RepoMan is lost (Fig. 5E). Overex-pression of these fragments does not affect cell cycle distri-bution (supplemental Fig. S2), and this result therefore sug-

gests that they can associate with R1A in interphase as wellas in mitosis. No differentially phosphorylated forms of PP2Acor B56! were identified in these experiments (data notshown).

Further Fragmentation of the C Terminus of RepoMan Re-veals Distinct Domains Mediating Association with RibosomalProteins, IPO7, the PP2A Complex and Proteins Related toDNA Damage Response—Similar to our concern that interac-tions with abundant importin proteins masked our ability toidentify lower abundance interactors in our nuclear RepoMan

FIG. 5. Mitosis-specific association of RepoMan/PP1 and PP2A/R1A/B56!. A, IP/WB analysis demonstrating copurification of B56! withendogenous RepoMan (RM) predominantly from extracts prepared from cells arrested in mitosis (using nocodazole or taxol), as compared withasynchronous cells or cells arrested in S-phase (using thymidine, hydroxyurea or L-mimosine). Lysate lanes demonstrating the relative amountsof RepoMan and B56! (and the loading control GAPDH) before IP are shown below the IP lanes. B, IP/WB analysis of endogenous RepoManimmunoprecipitated from either asynchronous or nocodazole (noc)-arrested cell extracts, compared with an IgG control IP. The WB wasprobed with the antibodies indicated. C, Quantitative comparison of GFP-PP1" and GFP-PP1! interactomes in nocodazole-arrested cells.Specific enrichment of RepoMan and PP2Ac/R1A/B56! with the ! isoform is indicated by high H:L and H:M ratios and low M:L ratios. PP1"is shown for comparison (high M:L ratio, and low H:L and H:M ratios). D, Phos-tag gel/WB analysis of R1A in asynchronous cell extracts versusextracts from cells arrested in early mitosis (using nocodazole or the Eg5 inhibitor STLC). The Phos-tag amplifies phosphorylation-inducedband shifts. E, Phos-tag gel/WB analysis of R1A copurified with FP-tagged wild type RepoMan, the RAXA mutant and the 305–713 and713–1023 fragments. Control GFP alone and RepoMan/713-1023 IPs are shown for comparison.



interactome screen, interaction of the C-terminal (496–1023)half of RepoMan with abundant ribosomal proteins has thepotential to mask lower abundance chromatin-associated in-teractors. Fluorescence imaging-based analysis indicatedthat chromatin association mapped predominantly to the713–1023 region (Fig. 2A), and we therefore used our quan-titative AP/MS approach (Fig. 3A) to map the fragmentomesof FP-RepoMan/496–713 and FP-RepoMan/713–1023. Thesamples were analyzed twice by LC-MS/MS, with a total of378 and 309 proteins identified and quantified, respectively.The top hits are summarized both in supplemental Tables S4,S5 and in the graph in Fig. 6A, which plots log H:L ratios(enrichment with 713–1023) versus log M:L ratios (enrichmentwith 496–713). Interaction with the PP2A complex (PP2Ac,R1A, and B56!) mapped to the 496–713 domain of RepoMan,although specificity for the B56 subunit isoform appeared tobe lost, with additional enrichment of B56", B56#, and B56$

(supplemental Table S4). This could either reflect increasedsensitivity of detection or indicate that additional regions ofRepoMan are required to confer specificity for a particularPP2A complex.

Association of ribosomal proteins with RepoMan alsomapped to the 496–713 region, with an even more dramaticshift of the isotope ratios of these proteins away from thecontaminants peak in the FP-RepoMan/496–713 IP (Fig. 3C).Fig. 6A and supplemental Table S4 also highlight the fact thatribosomal proteins represent a large fraction of the proteinsenriched with FP-RepoMan/496–713. Interaction with ribo-somal proteins was further mapped to the minimal 496–591domain (Fig. 6C, supplemental Fig. S5A), which, like the 496–713 fragment (Fig. 6A), is predominantly nucleoplasmic withadditional accumulation in nucleoli (supplemental Fig. S5C).The nucleolar pool accounts for !20% of the total nuclearpool of FP-RepoMan/496–591, as measured by quantitativeWB analysis of subcellular fractions (supplemental Fig. S5C).To demonstrate that increased nucleolar accumulation alonecannot account for increased enrichment of ribosomal pro-teins, we confirmed that both FP-RepoMan/496–591 and FP-RepoMan enrich significantly more RPL7 than either free FPor FP tagged with a short NLS (RKKR) that leads to accumu-lation in nucleoli (supplemental Figs. S5A, 5B).

Although full-length RepoMan does not show a steady-state accumulation in nucleoli (Fig. 2A and supplemental Fig.S4A), it does traffic through them (supplemental Fig. S5D),and therefore has the potential to come in contact with ribo-somal proteins either in the nucleoplasm, the nucleolus orboth. IP/WB results showing that RPL7 copurifies predomi-nantly with the nucleoplasmic pool of FP-RM/496–591 (sup-plemental Fig. S5C), suggest that the interaction occurs pri-marily in this region.

Another RepoMan interactor that mapped to the 496–713fragment is IPO7 (Fig. 6A, supplemental Table S4). As previ-ously noted, IPO7 binds full-length RepoMan most efficiently,and the SILAC IP results and weak binding observed for the

fragments in IP/WB experiments suggest that both N- andC-terminal domains are important for this binding. Havingobserved a weak interaction of IPO7 with both the 1–135 and496–591 fragments of RepoMan in IP/WB experiments (datanot shown), we tested binding to a 1–591 fragment that in-cludes both these domains. Increased enrichment of IPO7with this fragment (Fig. 6E) confirms that these two regionsare required for efficient binding, and explains the loss ofbinding when the protein is split in half at residue 496.

Although association of ribosomal proteins with RepoMancould be mapped to the predicted globular domain spanningresidues 496–591, association with the PP2A complex waslost when RepoMan was split in half at residue 591 (Fig. 6D).Phosphorylation of residue S591 in nocodazole-arrested cellshas been mapped both by our lab (Fig. 7A) and others (23, 24),and we therefore tested whether the phospho-status of thisresidue could affect association of RepoMan with the PP2Acomplex. As shown in supplemental Fig. S4G, there is nosignificant difference in the amount of PP2A complex mem-bers copurified with a nonphosphorylatable (S591A) or phos-phomimic (S591D) mutant compared with full-length FP-RepoMan. It is more likely that splitting at this particular sitedisrupts binding properties, as the C-terminal 591–1023 frag-ment showed very different nuclear retention and dynamicscompared with other C-terminal fragments (supplementalFigs. S1A, S1B).

The most striking observation for the 713–1023 fragmen-tome was the significant enrichment of four 14-3-3 proteinfamily members (Fig. 6A, supplemental Table S5), known tobind and regulate various chromatin-associated proteins. Thisassociation was validated for 14-3-3% by co-IP/WB analysis(Fig. 6F). The extreme C terminus of RepoMan (residues 946–1023) contains two predicted 14-3-3 binding sites (Fig. 8B).We observed that 14-3-3 protein binding is lost when thisregion is deleted from FP-RepoMan/713-1023, suggestingthat it is involved in the interaction (Fig. 6H).

Although we could validate the interaction of 14-3-3% withFP-RepoMan/713–1023 , we could not detect copurificationof 14-3-3 proteins with full-length FP-RepoMan. It is not yetclear whether this is a sensitivity issue, or whether the inter-action normally occurs with full-length RepoMan under veryspecific conditions that we have not yet identified. Theseproteins have been shown to bind nonspecifically to affinitymatrices (9), which can complicate validation. Direct compar-ison of the distribution of identified proteins between the N-(1–496) and C-terminal (496–1023) halves of RepoMan in theinitial fragmentome experiment revealed that 14-3-3 proteins,like histones, are slightly enriched with the C-terminal frag-ment, albeit buried in the threshold (supplemental Fig. S3C).This suggests that sensitivity of detection of specific bindingabove nonspecific background binding may be a factor in ourinability to validate the interaction with full-length RepoMan.

Another chromatin-associated protein found to be enrichedwith the 713–1023 fragment was PRKDC, the catalytic subunit



FIG. 6. C-terminal half of RepoMan contains distinct bindings domains for diverse interactors. A, Plot of log H:L ratio (enrichment inGFP-RepoMan/713–1023 IP) versus log M:L ratio (enrichment in GFP-RepoMan/496–713 IP). Contaminants cluster around a log ratio 0 (SILACratio 1:1). The 496–713 fragment (blue circle) enriched a cluster of ribosomal proteins (RPLs, dark pink triangles; RPSs, light pink triangles),PP2A complex members (orange squares) and importins (yellow diamonds), whereas the 713–1023 fragment (red circle) enriched several14-3-3 proteins and the DNA-dependent protein kinase PRKDC (green squares). Inset images are single optical sections of live HeLa cellsexpressing FP-RepoMan/496–713 or FP-RepoMan/713–1023. Both fragments are predominantly nuclear, with 496–713 showing additionalaccumulation within nucleoli. Scale bars are 5 !M. B, Validation of the mapping of PP2Ac association to the 496–713 domain. C, Interactionof the ribosomal protein RPL7 with the 496–713 domain of RepoMan was validated, and binding further mapped to the 496–591 domain. D,RPL7 and B56" associate equally with wild type RepoMan (RM) and the non-PP1 binding RAXA mutant. Although RPL7 association mappedto the 496–591 domain, splitting the C terminus of RepoMan at this site disrupts association with PP2A complex members. E, Consistent withthe weak interaction observed for IPO7 with regions in both the N- (1–135) and C- (496–1023, 496–591) terminus, the 1–591 fragment thatspans both of these regions copurifies IPO7 nearly as efficiently as full-length FP-RepoMan. F, Validation of the domain-specific interactionsof R1A (496–713), PRKDC (713–1023) and 14-3-3# (713–1023). G, Validation of the interaction of PRKDC with both the 713–1023 fragment andwith full-length FP-RepoMan. H, Validation of the interaction of 14-3-3# with the 713–1023 fragment. Removal of the extreme C terminus(947–1023) from this fragment abrogated 14-3-3# binding. Anti-FP antibodies were used to demonstrate the amount of fusion protein (or freeFP) recovered in each IP.



of the protein kinase DNA-PK. Although this protein is knownto bind nonspecifically to affinity matrices (9), we demon-strated that it is enriched above background with both fulllength FP-RepoMan and the 713–1023 fragment (Figs. 6F,6G).

We did not observe significant enrichment of histonesabove background in any of our SILAC IP experiments, how-ever they did show a slight enrichment with the 496–1023fragment (Fig. 4A, supplemental Fig. S3C). We carried out aseries of IP/WBs of full-length RepoMan and the fragmentsused in our SILAC experiments, assessing copurification

of histone H3 (supplemental Fig. S5E), which is a knownRepoMan/PP1 substrate (4). The results, summarized insupplemental Fig. S5F, indicate that although H3 is not sig-nificantly enriched above background with full-length FP-Re-poMan, significant enrichment is observed with both the 496–1023 and the 713–1023 fragments. This increased histoneassociation may represent a direct interaction and/or a higherdegree of association of RepoMan with chromatin complexesthat include histones. Interestingly, the histone H1 family, andH1X in particular, were enriched with the 496–713 fragment(supplemental Table S4), and we validated this increased asso-

FIG. 7. Effect of early mitotic phos-phorylation on RepoMan dynamicsand PP1 binding. A, Diagram noting thelocation of 11 phosphoresidues mappedin endogenous RepoMan immunopre-cipitated from nocodazole-arrestedcells. Pale gray boxes indicate the posi-tions of predicted NLSs whereas thedark gray box marks the PP1-bindingRVxF motif. B, Summary of FRAP exper-iments comparing the recovery rates ofsingle phosphomimic mutants (S/T toD/E) to that of wild type RepoMan. Adouble phosphomimic mutant of the twosites (S756E/S936D) mapped in thechromatin-associated 713–1023 domainwas also tested. Data are mean ! S.E.(n " 5–25). C, IP/WB experiment dem-onstrating the loss of PP1 binding in-duced by the RAXA and T412D phos-phomimic mutations compared with wildtype FP-RepoMan (FP-RM). The T412Anonphosphorylatable mutant retainsPP1 binding, as does the S401D phos-phomimic mutant. The results of 4 quan-tified experiments are summarized in (D).Data are mean ! S.E. Asterisks indicatea significant reduction in associated PP1compared with wild type FP-RepoMan(p # 0.05 in an unpaired Student’s t test).E, Endogenous RepoMan (RM) copuri-fies with FP-tagged PP1! from HeLa/GFP-PP1! cell extracts (both asynchro-nous and arrested in early mitosis usingnocodazole or taxol). F, EndogenousRepoMan copurifies with endogenousPP1! immunopurified from both asyn-chronous and nocodazole-arrested HeLacell extracts.



ciation by IP/WB analysis for both the 496–713 and 496–1023fragments (data not shown). Although the specific function of thisH1 variant is not yet clear, it was shown to exhibit a G1 phase-dependent nucleolar accumulation (25), which may relate to itsassociation with the nucleolar-targeted 496–713 fragment.

Other putative RepoMan interactors that were detectedclose to threshold in our fragmentome experiments and vali-dated by IP/WB analysis include Nucleolin (NCL), Caprin,G3BP2, hnRNP K, and hnRNP A1. Fig. 8 summarizes thevalidation experiments that were carried out for these andother interactors identified in the RepoMan fragmentome ex-periments, and the minimal association domains to whichthey were mapped.

Effects of Mitotic Phosphorylation of RepoMan on TurnoverDynamics and Protein Binding—Having previously observed ahigh level of RepoMan phosphorylation in early mitosis, coin-cident with release of the protein from chromatin (2), we setout to identify the specific residues by phosphopeptide map-ping to test the effect of mimicking phosphorylation (usingS/T - D/E phosphomimic mutants) on localization and turn-over dynamics in interphase cells. Endogenous RepoMan wasaffinity purified from nocodazole-arrested HeLa cell extracts,separated by SDS-PAGE and trypsin-digested, and phospho-peptides enriched using IMAC resin. Mass spectrometricanalysis identified 11 phosphopeptides (see SupplementalData set), highlighted on the RepoMan diagram in Fig. 7A.Most have also been identified in one or more large-scalephosphoproteomic screens (23, 24, 26–30), although this isthe first detection of phospho-S240. We mutated each ofthese residues singly to acidic D or E residues and comparedtheir turnover dynamics to that of wild-type RepoMan byFRAP analysis. Our prediction was that phosphorylation ofone or more of these sites would increase the turnover dy-namics of nucleoplasmic RepoMan by weakening associationwith chromatin. None of the single phosphomutants in-creased the turnover rate significantly (Fig. 7B), nor did adouble phosphomutant mimicking phosphorylation of bothresidues (S756 and S936) mapped in the C-terminal 713–1023domain. It is therefore unlikely that phosphorylation of Repo-Man at the onset of mitosis, in the region that we have shownto mediate interphase chromatin association, is sufficient toinduce release from chromatin.

A previous study had mapped T412 as the predominant sitephosphorylated by CDK1 in vitro, and suggested that thisphosphorylation results in a loss of PP1 binding in early mi-tosis (5). As this was inconsistent with our evidence, from bothRepoMan and PP1 interactome studies, that RepoMan/PP1

FIG. 8. Validation summary and model of the modular interac-tion events mediated by specific domains of RepoMan. A, Tablesummarizing the validation of the RepoMan interactors discussed inthis study. Where indicated, endogenous interactors were detected inpulldowns of full-length FP-tagged RepoMan (Co-IPs with FP-RM),endogenous RepoMan was detected in pulldowns of FP-tagged in-teractors (RM co-IPs with FP-Protein) and/or endogenous interactorswere detected in pulldowns of endogenous RepoMan (Co-IPs withendog RM). With the exception of 14-3-3!, all of the interactorsshown here validated with full-length RepoMan. B, Diagram summa-rizing the domain-specificity of the interactions uncovered using the“fragmentome” mapping approach. Although the extreme N terminusmediates most interactions with importins and NUPs, IPO7 (and othernonclassic importins) binds weakly to both the 1–135 and 496–591regions of RepoMan, and more efficiently to the 1–591 fragmentencompassing both of these regions. The PP1-binding RVxF domainin the N terminus mediates interaction with PP1" and PP1#. Region496–591, which mediates interaction with ribosomal proteins andshows additional accumulation in nucleoli, contains both a predictedbipartite nuclear localization signal (NLS, underlined) and a predictednucleolar localization signal (NoLS, bold text). The longer 496–713domain is required for association with PP2Ac/R1A/B56#, and thisassociation occurs predominantly in early mitosis. The chromatin

localization pattern observed for RepoMan in interphase cells is main-tained by the extreme C terminus (713–1023), which also mediatesassociation with histone H3 and with the DNA damage response-related PRKDC kinase and 14-3-3 proteins. Deletion of the extreme Cterminus (947–1023), which contains 2 predicted 14-3-3 binding sites,results in loss of 14-3-3 binding.



complexes persist in cells arrested in early mitosis (Figs. 5B,7E, 7F), we directly assessed the interaction of endogenousPP1 with FP-tagged RepoMan/T412 mutants. As shown inFig. 7C, mimicking phosphorylation at this site with a T412Dmutant resulted in a significant, but not complete, loss of PP1binding, whereas our previously described RAXA mutant (1)abolished PP1 binding entirely. The nonphosphorylatableT412A mutant retained PP1 binding, as did the S401D mutant(which is even closer to the PP1 binding motif found at resi-dues 392–395). This experiment (with the exception of theT412A mutant) was repeated three times and the resultssummarized in Fig. 7D. The persistence of a RepoMan/PP1complex in early mitosis is likely because of two factors: 1. theincomplete loss of PP1 binding when this site is phosphoryl-ated and 2. the existence of a pool of RepoMan that is notphosphorylated at this site. In support of the latter, we foundthat a relatively efficient depletion (!70%) of endogenousPP1! from nocodazole-arrested cells copurified RepoMan buthad only a minimal effect on the total level of RepoMan in theextract (Fig. 7F). Although this could be accounted for in partby RepoMan persisting in complexes with the " isoform ofPP1, it also suggests that there are pools of RepoMan in thecell that are bound to PP1 and pools that are not bound toPP1. Because nobody has yet quantified the total amount ofRepoMan that is phosphorylated on T412 (or any of the otherresidues), we can assume that there is a mixed population inthe cell. Phosphorylation of this residue was also detected inasynchronous T cells in which the ERK pathway was inhibited(26), suggesting that it is not necessarily mitosis-specific.

DISCUSSION

We initiated this study with the intention of mapping inter-phase chromatin-associated RepoMan complexes, usingtransiently expressed FP-tagged fusion protein as bait in ouroptimized quantitative SILAC IP workflow (9). Although it ispossible to affinity purify endogenous RepoMan using ourin-house or commercially available polyclonal antibodies, thedepletion efficiency varies. More importantly, all of these an-tibodies detect unrelated bands in cell lysates in addition toRepoMan, suggesting a high potential for cross-reactivity thatwould complicate a nonbiased AP/MS screen (10). We con-firmed this by attempting such a screen with the most com-monly used commercial RepoMan antibody and identifying/validating strong cross-reactivity with at least one othercellular protein (DBC1/KIAA1967; data not shown). Althoughour endogenous RepoMan interactome screen did identifyseveral top hits that we later identified/validated in our taggedRepoMan screens (PP1" and !, KPNB1, KPNA4, Caprin,G3BP2, hnRNP K and several ribosomal proteins), other hitsdid not validate as RepoMan interactors, suggesting copuri-fication with DBC1 or another protein with which the antibodycross-reacted.

In contrast, FP-tagged proteins can be rapidly and repro-ducibly purified from extracts using high affinity/specificity

single chain llama antibodies, and the powerful combinationof fluorescence imaging with quantitative proteomics hasbeen used by our lab and others to dissect diverse cellularpathways including protein phosphatase targeting (1), ribo-somal protein dynamics (12), and composition of the ana-phase promoting complex (31, 9). When applied here to theanalysis of FP-tagged RepoMan complexes in the nucleus,we identified and validated a handful of known (PP1, KPNB1,KPNA2, NUP153) and novel (KPNA4, IPO7, PP2R1A) interac-tors, all of which (with the exception of PP1) were enrichedequally with the wild type protein and non-PP1-binding RAXAmutant. Detection of a RepoMan/R1A/PP2A complex wasparticularly intriguing because it suggested a stable associa-tion of two different serine/threonine phosphatase complexes.When the increased sensitivity of our fragmentome approachuncovered an additional member of this PP2A complex, theregulatory subunit B56!, we explored the interaction furtherand discovered that it predominantly occurs between theendogenous proteins in mitosis. This was consistent with ourobserved copurification of RepoMan, R1A, and B56! withFP-PP1! from nocodazole-arrested HeLa/GFP-PP1! cell ex-tracts. Interestingly, we and others have shown phosphory-lation of RepoMan on residue T412 (which can affect interac-tion with PP1) in early mitosis, although the stoichiometry ofthis phosphorylation has not yet been quantified. We demon-strate here that mimicking phosphorylation at this site dis-rupts but not does completely abrogate PP1 binding. Takentogether with our finding that a substantial fraction ofRepoMan remains following near complete depletion of en-dogenous PP1! from nocodazole-arrested cell extracts, wesuggest that RepoMan is present in more than one complexunder these conditions (i.e. with and without PP1), makingmitotic regulation of this particular PP1 complex more com-plicated than previously thought (5).

B56-PP2A complexes have been implicated both in theprotection of centromeric cohesion before the metaphase-anaphase transition (32) and in the regulation of the stableattachment of kinetochores and microtubules (33). Althoughseveral distinct roles for RepoMan/PP1 have been identifiedat mitotic exit, including counteraction of Aurora B activity onanaphase kinetochores to stabilize chromosome segregation(3) and coordination of nuclear envelope reassembly (5), thecomplex has also been shown to oppose the Haspin kinase-mediated spreading of histone H3T3 phosphorylation to chro-mosome arms before metaphase, before mediating net de-phosphorylation of this residue at mitotic exit (4). Futurestudies will address the functional significance of the PP1/PP2A complex association that we identified here.

We found no evidence that RepoMan/PP1 regulates netphosphorylation of any of the PP2A complex subunits (orvice-versa), although we did demonstrate a previously unre-ported decrease in net R1A phosphorylation in cells enteringmitosis. This was consistent with our observation thatRepoMan, which interacts with R1A predominantly in mitosis,



associates with the less phosphorylated form of the protein.Interestingly, although overexpression of smaller fragments ofRepoMan (305–713 and 496–713) that bind R1A had no effecton cell cycle distribution (i.e., did not increase the fraction ofcells in mitosis), these fragments were able to copurify boththe low/unphosphorylated (mitosis) and the more phosphor-ylated (interphase) forms of the protein. This suggests thatadditional regions in RepoMan mediate the mitotic specificityof the interaction.

We also noted a loss of B56 isoform specificity when wescreened the 496–1023 C-terminal half of RepoMan versusthe smaller 496–713 region, with the latter enriching not onlyB56!, but also B56", #, and $. We do not know yet whetherthis is simply a detection issue, or whether it suggests thatadditional regions in RepoMan contribute to binding specific-ity. Mass spectrometric analysis of the protein Shugoshin,shown to associate with PP2Ac/B56, also detected interac-tion with numerous isoforms of the B56 subunit (32), suggest-ing that they may be interchangeable in certain complexes orunder certain conditions. In addition, a recent study that ap-peared while this work was under review detected copurifi-cation of RepoMan/CDCA2 with tagged R1A (confirming ourfindings) and with both tagged B56! and B56" (34). Althoughthe only interactions that have been confirmed between theendogenous proteins so far are the copurifications of PP2Ac,R1A, and B56! with RepoMan presented here, we cannot ruleout the possibility that other B56 isoforms interact withRepoMan in vivo.

Another surprising finding uncovered by our fragmentomeanalysis is the presence of a functional nucleolar localizationsignal within RepoMan. We have shown here that althoughRepoMan does not accumulate within this nuclear structureunder steady-state conditions, it does traffic through it. Thistrafficking may be related to the equally surprising finding thatRepoMan associates with ribosomal proteins, which we wentto great lengths in this study to rigorously validate. Theseabundant proteins are common contaminants in AP/MS ex-periments, however it is important in a nonbiased screen tonot simply subtract them from data sets because there arecircumstances in which they are genuinely enriched with theprotein of interest (13). When our fragmentome screens re-vealed significant enrichment of ribosomal proteins with theC-terminal half of RepoMan (further mapped to the 496–713and then 496–591 domain), we set out to validate interactionwith full-length RepoMan, demonstrating significant enrich-ment with both the FP-tagged and endogenous protein. Thishighlights the power of the fragmentome approach, which canuncover interactions hidden by the lower sensitivity and sig-nal-to-noise ratio of more complex full-length protein interac-tome screens.

Taken together with our data showing that the same do-main within RepoMan mediates association with numerousimport factors involved in a range of ribosomal protein importpathways (IPO7, IPO5, KPNB2, KPNB1, and IPO4), interac-

tion with ribosomal proteins suggests the possibility of a rolefor RepoMan in regulation of ribosome biogenesis. WithRepoMan found at the nuclear pore complex, in the nucleo-plasm, and trafficking through nucleoli, it is not unfeasible tosuggest some type of chaperone role in nuclear import and/ornucleolar targeting of ribosomal proteins. There is a wealth ofliterature linking reversible phosphorylation events to regula-tion of nucleocytoplasmic transport (see (35) for review), in-cluding enhanced recognition or binding affinity for importinson phosphorylation of residues within (36) or near (37) classicNLSs. Phosphorylation events have also been shown to acti-vate noncanonical transport signals mediating nuclear import,as in the case of enhanced recognition of phosphorylatedERK1/2 by IPO7. Nuclear accumulation of active ERK5 istriggered when phosphorylation unmasks an NLS by disrupt-ing an NES (38), the consequences of which can includeincreased activation of the AP-1 transcription factor that con-trols proliferation, differentiation and apoptosis (39). Treat-ment with the PP1/PP2A inhibitors okadaic acid and micro-cystin has been shown to inhibit nuclear import pathwaysmediated by KPNB1 and KPNB2/transportin (40), whereas inyeast, PP1 activity has been shown to antagonize PKA-de-pendent phosphorylation at the nuclear import domain ofMsn2, a transcription factor involved in stress response (41).

Although IPO7 can act as an adaptor-like protein in asso-ciation with KPNB1, mediating nuclear import of proteinssuch as histone H1 (another protein enriched with the 496–713 fragment), it can also function as an autonomous nucleartransport receptor. With respect to this latter role it has beenshown, along with IPO5 and KPNB2, to mediate nuclear im-port of ribosomal proteins including RPL23A, RPS7, andRPL5 (42). Assembly of the 40S and 60S ribosomal subunitstake place in the nucleolus, and the !75 ribosomal proteinsrequired for this assembly and maturation that are synthe-sized in the cytoplasm are rapidly and actively imported intothe nucleus via diverse import pathways (43) and targeted tothe nucleolus (12). The redundancy of import pathways usedfor ribosomal proteins is likely required to support the highproduction rate of ribosomes in proliferating cells, but com-plicates regulation of import. A regulatory mechanism linkedto all of these pathways would therefore provide a means ofrapidly switching import on or off as required. We will continueto explore the potential for such a regulatory role for RepoManin ribosomal protein transport, and whether it is mediated viaPP1.

With respect to chromatin association, we showed that theextreme C terminus of RepoMan (residues 713–1023) confersthe predominant interphase chromatin localization pattern,although its more rapid turnover dynamics compared withfull-length RepoMan and the larger 496–1023 C-terminal halfsuggest that certain binding events may have been disruptedor weakened. Of the top hits in the FP-RepoMan/713-1023fragmentome, several have been linked to DNA damage re-sponse pathways, including the 14-3-3 proteins gamma, ep-



silon, zeta, and theta (for review see (44), the catalytic subunitof the DNA damage sensor kinase DNA-PK (PRKDC; 45), theDNA mismatch repair protein MSH6 (46) and TRIP13, which isinvolved in DNA double-strand break repair (47).

PRKDC, which we confirmed copurifies with full-length FP-RepoMan, had previously been shown to interact with bothPP2A and the PP2A-like protein phosphatase 6 (PP6;48).PRKDC recruits the PP6 complex to sites of DNA damage,where it dephosphorylates !-H2AX. This results in dissolutionof ! irradiation-induced foci and release of cells from theG2/M checkpoint. A PRKDC/RepoMan complex would likelyfeed into the damage sensing/repair pathway at anotherpoint. Although previous work by another lab has shown anassociation between RepoMan and the ATM kinase (8), wedid not identify ATM in our interactome screens and could notdetect an interaction by IP/WB analysis of endogenousRepoMan, full-length FP-RepoMan or any of our fragments.This may be because of differences in cell lines or experimen-tal conditions.

In summary, by employing fluorescence imaging tech-niques to monitor localization, nuclear retention and turnoverdynamics of specific regions of RepoMan, we identified spe-cific domains involved in association with the nuclear porecomplex, trafficking through nucleoli and association withchromatin. We then directly extended these observations, viaquantitative interactome screens, to identify the underlyingprotein-protein binding events. The results highlight the factthat RepoMan is a modular protein that participates in morethan one regulatory pathway and likely exists in multiple com-plexes throughout the cell at any given time. Importantly, thispowerful technique can be extended to other modular pro-teins to add both increased sensitivity of detection and do-main specificity to interactome analyses.

Acknowledgments—We thank Lawrence Puente at the OttawaHospital Research Institute Proteomics Core Facility for technicalsupport. We also thank Dr. Judith Sleeman for assistance with FRAPanalysis and Drs. Jocelyn Cote, Anne-Claude Gingras, Saskia Hutten,Angus Lamond and Ben Wang for providing plasmids and antibodiesused in this study.

* This work was supported by a Canadian Cancer Research/TerryFox Foundation New Investigator Award (Ref: 20148). LTM holds aCIHR New Investigator Salary Support Award.

!S This article contains supplemental Tables S1 to S5 and Figs. S1to S9.

! To whom correspondence should be addressed: Department ofCellular and Molecular Medicine, Ottawa Institute of Systems Biology,Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa,ON K1H 8M5, Canada. Tel.: 1-613-562-5800 ! 8068; Fax: 1-613-562-5636; E-mail: [email protected].

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“fragmentome” mapping reveals novel, domain-specific partners

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